The invention relates to the design of carbohydrate processing inhibitors for glycosyltransferases based on the conformational properties of the sugar phosphate linkage and/or phosphate linkages in nucleotide-sugar donors for the glycosyltransferases.
The oligosaccharide chains of Nxe2x80x94 and O-linked glycoproteins play a crucial role in a number of biological processes [1,2]. Their biosynthesis and degradation pathways are therefore areas of significant interest for biology, medicine, and biotechnology. The assembly of the various types of oligosaccharides involves several glycosidases and glycosyltransferases. In comparison with glycosidases, the mechanisms of which have been characterized in some detail [3-5] the catalytic mechanism of glycosyltransferases have not yet been investigated in detail, though some kinetic studies have been reported [6-18].
Glycosyltransferases are a diverse group of enzymes that catalyze the transfer of a single monosaccharide unit from a donor to the hydroxyl group of an acceptor saccharide. [19, 20] The acceptor can be either a free saccharide, glycoprotein, glycolipid, or polysaccharide. The donor can be a nucleotide-sugar, dolichol-phosphate-sugar or dolichol-pyrophosphate-oligosaccharide. Glycosyltransferases show a precise specificity for both the acceptor and sugar donor and generally require the presence of a metal cofactor, usually a divalent cation like manganese.
The knowledge of the structure of nucleotide-sugars is prerequisite for understanding the catalytic mechanism of glycosyltransferases and for developing inhibitors for these enzymes. The 3-D structure of nucleotide-sugars is determined to some extent by the conformation adopted by the phosphate linkage. However, despite the importance of the conformation adopted by the diphosphate linkages on the overall shape of nucleotide-sugars, the structure and the conformational properties of such linkage remains somewhat unspecified.
Few calculations have been performed on the diphosphate linkage. The structure of the lowest energy conformers of pyrophosphoric acid, its anions and alkali salts were studied to model the hydrolysis of pyrophosphate [2-5]. The presence of hydroxyl groups in pyrophosphates stabilizes their lowest energy conformers by intramolecular hydrogen bonds. However, such stabilizing interactions are not possible in nucleotide-sugars and the diphosphate linkage in nucleotide-sugars should exhibit a different conformational behavior.
The invention relates to the design of carbohydrate processing inhibitors for glycosyltransferases based on the conformational properties of the sugar-phosphate linkage and/or phosphate linkage in sugar nucleotide donors for the glycosyltransferases. The method permits the identification early in the drug development cycle of compounds which have advantageous properties.
In particular, the present inventors studied the conformational properties of the sugar-phosphate linkage with ab initio methods using the 2-O-methylphosphono-tetrahydropyran anion (1 in FIG. 1) and sodium 2-O-methylphosphono-tetrahydropyran (2 in FIG. 1) as models. The ab initio energy and geometry of the conformers around the C1xe2x80x94O1 and Oxe2x80x94P bonds were determined at various levels of the self-consistent field (SCF) and adiabatic connection method of density functional theory. At all levels of ab initio theory, compound 1 preferred the trans to the gauche conformer around the C1xe2x80x94O1 bond. The presence of a sodium counter-ion completely reverses the relative energy of the conformers, such that in the ion-pair complex 2, the gauche conformer about the C1xe2x80x94O1 bond is favored
The present inventors also carried out an ab initio study of the sugar-diphosphate linkage. Ab initio molecular orbital calculations of the 2-O-methyldiphosphono-tetrahydropyran dianion and the magnesium 2-O-methyldiphosphono-tetrahydropyran were used to model the conformational behaviour of the sugar-diphosphate linkage in sugar-nucleotides. The geometry and energy of conformers were calculated at different basis set levels, from 6-31G* to cc-pVTZ(xe2x88x92f)++, using the SCF, DFT/B3LYP, and LMP2 methods. The vibrational frequencies were calculated at the HF/6-31G* level and the zero-point energy, thermal and entropy corrections were evaluated. The results of conformational analyses show that interactions of the diphosphate linkage with the Mg2+ cation alter the conformational preferences about the anomeric and the diphosphate linkages. These changes influence the overall 3D-shape adopted by nucleotide-sugars.
The differences in structures with the ions indicates an important function of the metal cofactor in the catalytic mechanism of glycosyltransferases. Complexation of the phosphate with the metal ion changes the conformation about the phosphate linkages and more specifically about one of the Pxe2x80x94O bonds going from gauche to trans orientation; it changes the conformation of the sugar-phosphate linkage from trans to gauche orientation; it influences the overall 3D-shape adopted by molecules containing phosphate linkages such as sugar donors in order to adopt a correct shape for optimal enzymatic recognition and to achieve maximal catalytic efficiency; it activates the sugar-oxygen glycosidic bond by elongating the sugar-oxygen bond; and/or, it changes the charge distribution to make the protonation of the glycosidic oxygen more favored.
Therefore broadly stated, the present invention relates to a method for preparing a potential inhibitor of a glycosyltransferase comprising:
(a) combining a first sugar, a phosphate group, and a second sugar that is transferred by the glycosyltransferase to an acceptor for the glycosyltransferase, under conditions appropriate for formation of a bond between a carbon atom of the first sugar and a first oxygen atom of the phosphate group, and formation of a linkage between a carbon atom of the second sugar and a second oxygen atom of the phosphate group, wherein the orientation of the linkage is antiperiplanar, and preferably the distance between the carbon atom linked to the first sugar and the carbon atom linked to the second sugar is in the range 3.7 xc3x85 to 4.2 xc3x85;
(b) combining a first sugar, a phosphate group, an ion, and a second sugar that is transferred by the glycosyltransferase to an acceptor for the glycosyltransferase, under conditions appropriate for formation of a bond between a carbon atom of the first sugar and a first oxygen atom of the phosphate group, a linkage between a carbon atom of the second sugar and a second oxygen atom of the phosphate group, and an electrostatic interaction between free oxygen atoms of the phosphate group and the ion, and wherein the orientation of the linkage is synclinal, and, preferably the distance between the carbon atom linked to the first sugar and the carbon atom linked to the second sugar is in the range 3.7 xc3x85 to 4.5 xc3x85;
(c) combining a first sugar, a diphosphate group, and a second sugar that is transferred by the glycosyltransferase to an acceptor for the glycosyltransferase, under conditions appropriate for formation of a bond between a carbon atom of the first sugar and an oxygen atom of a first phosphate of the diphosphate group, and formation of a linkage between a carbon atom of the second sugar and an oxygen atom of a second phosphate of the diphosphate group, wherein the orientation of the linkage is antiperiplanar, phosphorous-oxygen bonds linking the first phosphate to the second phosphate of the diphosphate group are in a synclinal or anticlinal orientation, and synclinal orientation, respectively, or symmetrically related orientation, and preferably the distance between the carbon atom linked to the first sugar and the carbon atom linked to the second sugar is in the range 4.9 xc3x85 to 5.3 xc3x85; or
(d) combining a first sugar, a diphosphate group, an ion, and a second sugar that is transferred by the glycosyltransferase to an acceptor for the glycosyltransferase, under conditions appropriate for formation of a bond between a carbon atom of the first sugar and an oxygen atom of a first phosphate of the diphosphate group, a linkage between a carbon atom of the second sugar and an oxygen atom of a second phosphate of the diphosphate group, and an electrostatic interaction between two or more, preferably three, oxygen atoms of the diphosphate group and the ion, and wherein the orientation of the linkage is synclinal, phosphorous-oxygen bonds linking the first phosphate to the second phosphate of the diphosphate group are in antiperiplanar or -anticlinal orientation, and synclinal orientation, respectively, or symmetrically related orientations, and preferably the distance between the carbon atom linked to the first sugar and the carbon atom linked to the second sugar is in the range 5.1 xc3x85 to 5.8 xc3x85.
The method of the invention may be a computer-implemented method for designing potential inhibitors of a glycosyltransferase. The method may comprise one of the following:
A. designing a nucleotide-sugar with a monophosphate linkage by (a) selecting a molecule comprising a first sugar, a phosphate group, and a second sugar that is transferred by the glycosyltransferase to an acceptor for the glycosyltransferase, wherein there is a bond between a carbon atom of the first sugar and a first oxygen atom of the phosphate group, and a linkage between a carbon atom of the second sugar and a second oxygen atom of the phosphate group, (b) optimizing the conformation of the molecule using ab initio quantum chemistry methods so that the orientation of the linkage is antiperiplanar, and preferably the distance between the carbon atom linked to the first sugar and the carbon atom linked to the second sugar is in the range 3.7 xc3x85 to 4.2 xc3x85;
B. designing a nucleotide-sugar with a monophosphate linkage and having an electrostatic interaction between free oxygen atoms of the monophosphate and an ion by (a) selecting a molecule comprising a first sugar, a phosphate group, an ion, and a second sugar that is transferred by the glycosyltransferase to an acceptor for the glycosyltransferase, wherein there is a bond between a carbon atom of the first sugar and a first oxygen atom of the phosphate group, a linkage between a carbon atom of the second sugar and a second oxygen atom of the phosphate group, and an electrostatic interaction between free oxygen atoms of the phosphate group and the ion, (b) optimizing the conformation of the molecule using ab initio quantum chemistry methods so that the orientation of the linkage is synclinal, and, preferably the distance between the carbon atom linked to the first sugar and the carbon atom linked to the second sugar is in the range 3.7 xc3x85 to 4.5 xc3x85;
C. designing a nucleotide-sugar with a diphosphate linkage by (a) selecting a molecule comprising a first sugar, a diphosphate group, and a second sugar that is transferred by the glycosyltransferase to an acceptor for the glycosyltransferase, and wherein there is a bond between a carbon atom of the first sugar and an oxygen atom of a first phosphate of the diphosphate group, and a linkage between a carbon atom of the second sugar and an oxygen atom of a second phosphate of the diphosphate group, and (b) optimizing the conformation of the molecule using ab initio quantum chemistry methods so that the orientation of the linkage is antiperiplanar, phosphorous-oxygen bonds linking the first phosphate to the second phosphate of the diphosphate group are in a synclinal or anticlinal orientation, and synclinal orientation, respectively, or symmetrically related orientations; and preferably the distance between the carbon atom linked to the first sugar and the carbon atom linked to the second sugar is in the range 4.9 xc3x85 to 5.3 xc3x85; or
D. designing a nucleotide-sugar with a diphosphate linkage and having an electrostatic interaction between free oxygen atoms of the diphosphate and an ion by (a) selecting a molecule comprising a first sugar, a diphosphate group, an ion, and a second sugar that is transferred by the glycosyltransferase to an acceptor for the glycosyltransferase, wherein there is a bond between a carbon atom of the first sugar and an oxygen atom of a first phosphate of the diphosphate group, a linkage between a carbon atom of the second sugar and an oxygen atom of a second phosphate of the diphosphate group, and an electrostatic interaction between two or more, preferably three, free oxygen atoms of the diphosphate group and the ion, (b) optimizing the conformation of the molecule using ab initio quantum chemistry methods so that the orientation of the linkage is synclinal, phosphorous-oxygen bonds linking the first phosphate to the second phosphate of the diphosphate group are in antiperiplanar or -anticlinal orientation, and synclinal orientation, respectively, or symmetrically related orientations, and preferably the distance between the carbon atom linked to the first sugar and the carbon atom linked to the second sugar is in the range 5.1 xc3x85 to 5.8 xc3x85.
The ab initio calculations may be carried out using commercially available ab initio computer programs (e.g. Gaussian, Gaussian, Inc. Pittsburgh, Pa., Jaguar, Schrodinger, Inc. Portland, Oreg., Turbomole 95.0 program, San Diego: Biosym/MSI, 1995) using standard basis sets, and optimization of the geometry may be performed at the SCF level with the 6-31G* basis set.
The invention also contemplates inhibitors obtained using the methods of the invention. In an embodiment a potential inhibitor of a glycosyltransferase is provided comprising:
(a) a first sugar, a phosphate group, and a second sugar that is transferred by the glycosyltransferase to an acceptor for the glycosyltransferase, wherein there is a bond between a carbon atom of the first sugar and a first oxygen atom of the phosphate group, and a linkage between a carbon atom of the second sugar and a second oxygen atom of the phosphate group, wherein the orientation of the linkage is antiperiplanar, and preferably the distance between the carbon atom linked to the first sugar and the carbon atom linked to the second sugar is in the range 3.7 xc3x85 to 4.2 xc3x85;
(b) a first sugar, a phosphate group, an ion, and a second sugar that is transferred by the glycosyltransferase to an acceptor for the glycosyltransferase, wherein there is a bond between a carbon atom of the first sugar and a first oxygen atom of the phosphate group, a linkage between a carbon atom of the second sugar and a second oxygen atom of the phosphate group, and an electrostatic interaction between free oxygen atoms of the phosphate and the ion, and wherein the orientation of the linkage is synclinal, and, preferably the distance between the carbon atom linked to the first sugar and the carbon atom linked to the second sugar is in the range 3.7 xc3x85 to 4.5 xc3x85;
(c) a first sugar, a diphosphate group, and a second sugar that is transferred by the glycosyltransferase to an acceptor for the glycosyltransferase, and wherein there is a bond between a carbon atom of the first sugar and an oxygen atom of a first phosphate of the diphosphate group, and a linkage between a carbon atom of the second sugar and an oxygen atom of a second phosphate of the diphosphate group, and wherein the orientation of the linkage is antiperiplanar, phosphorous-oxygen bonds linking the first phosphate to the second phosphate of the diphosphate group are in a synclinal or anticlinal orientation; and synclinal orientation, respectively, or symmetrically related orientations, and preferably the distance between the carbon atom linked to the first sugar and the carbon atom linked to the second sugar is in the range 4.9 xc3x85 to 5.3 xc3x85; or
(d) a first sugar, a diphosphate group, an ion, and a second sugar that is transferred by the glycosyltransferase to an acceptor for the glycosyltransferase, wherein there is a bond between a carbon atom of the first sugar and an oxygen atom of a first phosphate of the diphosphate group, a linkage between a carbon atom of the second sugar and an oxygen atom of a second phosphate of the diphosphate group, and an electrostatic interaction between two or more, preferably three, free oxygen atoms of the diphosphate group and the ion, wherein the orientation of the linkage is synclinal, phosphorous-oxygen bonds linking the first phosphate to the second phosphate of the diphosphate group are in -anticlinal or antipleriplanar orientation, and synclinal orientation, respectively, or symmetrically related orientations, and preferably the distance between the carbon atom linked to the first sugar and the carbon atom linked to the second sugar is in the range 5.1 xc3x85 to 5.8 xc3x85.
In a specific embodiment of the invention a computer-implemented method is provided for designing a potential inhibitor of a glycosyltransferase comprising
(a) selecting a molecule comprising a group of the formula I 
xe2x80x83wherein C7 forms part of a first sugar; C1 forms part of a second sugar that is transferred by the glycosyltransferase to an acceptor for the glycosyltransferase; "PHgr" is a dihedral angle defining rotation about C1xe2x80x94O1; xcexa81 is a dihedral angle defining orientation about O1xe2x80x94P3; xcexa82 is a dihedral angle defining orientation about P3xe2x80x94O4, xcexa83 is a dihedral angle defining orientation about O4xe2x80x94P5, and
(b) optimizing the conformation of the molecule so that "PHgr" is in an antiperiplanar orientation, xcexa82 is in a synclinal orientation or symmetrically related orientation, and xcexa83 is in a synclinal or anticlinal orientation or a symmetrically related orientation. In a more preferred embodiment xcexa82 is in a synclinal or -synclinal orientation and xcexa83 is in a anticlinal or -anticlinal orientation. Most preferably "PHgr" is between about 1000xc2x0 and 170xc2x0 or symmetrically related orientations, xcexa82 is between about 60xc2x0 and 120xc2x0 or symmetrically related orientations, and xcexa83 is between about xe2x88x9250xc2x0 and xe2x88x92130xc2x0 or symmetrically related orientations.
In an additional specific embodiment of the invention a computer-implemented method is provided for designing a potential inhibitor of a glycosyltransferase comprising
(a) selecting a molecule comprising a group of the formula I and an ion 
xe2x80x83wherein C7 forms part of a first sugar; C1 forms part of a second sugar that is transferred by the glycosyltransferase to an acceptor for the glycosyltransferase; "PHgr" is a dihedral angle defining rotation about C1xe2x80x94O1; xcexa81 is a dihedral angle defining orientation about O1xe2x80x94P3; xcexa82 is a dihedral angle defining orientation about P3xe2x80x94O4, and T3 is a dihedral angle defining orientation about O4xe2x80x94P5, and
(b) optimizing the conformation of the molecule so that there is an electrostatic interaction between two or more, preferably three, free oxygen atoms of the molecule of the formula I and the ion, "PHgr" is in a synclinal orientation, xcexa82 is in a synclinal or a symmetrically related orientation, xcexa83 is in an -anticlinal or antiperiplanar orientation or a symmetrically related orientation, more preferably xcexa82 is in a synclinal orientation and xcexa83 is in an -anticlinal orientation. In a most preferred embodiment "PHgr" is between about 40xc2x0 and 100xc2x0 or symmetrically related orientations, xcexa82 is between about 60xc2x0 and 110xc2x0 or symmetrically related orientations, and xcexa83 is between about xe2x88x9290xc2x0 and xe2x88x92100xc2x0 (-ac) or 180xc2x110xc2x0 (ap) or symmetrically related orientations.
The invention contemplates a glycosyltransferase inhibitor comprising a group of the formula I 
wherein C7 forms part of a first sugar; C1 forms part of a second sugar that is transferred by the glycosyltransferase to an acceptor for the glycosyltransferase; "PHgr" is a dihedral angle defining rotation about C1xe2x80x94O2; xcexa81 is a dihedral angle defining orientation about O1xe2x80x94P3; xcexa82 is a dihedral angle defining orientation about P3xe2x80x94O4, and xcexa83 is a dihedral angle defining orientation about O4xe2x80x94P5, and wherein "PHgr" is in an antipleriplanar orientation, xcexa82 is in a synclinal orientation or symmetrically related orientation, and xcexa83 is in a synclinal or anticlinal orientation or a symmetrically related orientation. In a more preferred embodiment xcexa82 is in a synclinal or -synclinal orientation and xcexa83 is in a anticlinal or -anticlinal orientation. Most preferably, "PHgr" is between about 100xc2x0 and 170xc2x0 or symmetrically related orientations, xcexa82 is between about 60xc2x0 and 120xc2x0 or symmetrically related orientations, and xcexa83 is between about xe2x88x9250xc2x0 and xe2x88x92130xc2x0, or symmetrically related orientations.
The invention contemplates a glycosyltransferase inhibitor comprising a group of the formula I in combination with an ion 
wherein C7 forms part of a first sugar, C1 forms part of a second sugar that is transferred by the glycosyltransferase to an acceptor for the glycosyltransferase, "PHgr" is a dihedral angle defining rotation about C1xe2x80x94O1; xcexa81 is a dihedral angle defining orientation about O1xe2x80x94P3; xcexa82 is a dihedral angle defining orientation about P3O4, and xcexa83 is a dihedral angle defining orientation about O4xe2x80x94P5, and wherein there is an electrostatic interaction between two or more, preferably three, free oxygen atoms of the molecule of the formula I and the ion, "PHgr" is in a synclinal orientation, xcexa82 is in a synclinal or a symmetrically related orientation, xcexa83 is in an -anticlinal or antiperplanar orientation or a symmetrically related orientation, more preferably xcexa82 is in a synclinal orientation and xcexa83 is in an -anticlinal orientation. In a most preferred embodiment "PHgr" is between about 40xc2x0 and 100xc2x0 or symmetrically related orientations, xcexa82 is between about 60xc2x0 and 110xc2x0 or symmetrically related orientations, and xcexa83 is between about xe2x88x9290xc2x0 and xe2x88x92100xc2x0 (xe2x88x92ac) or 180xc2x110xc2x0 (ap) or symmetrically related orientations.
Enzymes for which inhibitors may be prepared in accordance with the invention are glycosyltransferases including eukaryotic glycosyltransferases involved in the biosynthesis of glycoproteins, glycolipids, glycosylphosphatidylinositols and other complex glycoconjugates, and prokaryotic glycosyltransferases involved in the synthesis of carbohydrate structures of bacteria and viruses, including enzymes involved in LOS and lipopolysaccharide biosynthesis. Examples of glycosyltransferases are N-acetylglucosaminyltransferases, including N-acetylglucosaminyltransferases I through V, and xcex2-1,3-galactosyl-O-glycosyl-glycoprotein xcex21,6-N-acetylgucosaminyl transferase (core 2 GlcNAc). Table 16 provides examples of eukaryotic glycosyltransferases, and their sugar nucleotide donors and acceptors. A xe2x80x9csugar nucleotide donorxe2x80x9d refers to a nucleotide coupled to a selected sugar that is transferred by a glycosyltransferase to an acceptor. (The selected sugar is also referred to herein as xe2x80x9csecond sugarxe2x80x9d). An xe2x80x9cacceptorxe2x80x9d refers to the part of a carbohydrate structure (e.g. glycoprotein, glycolipid) where the selected sugar is transferred by the glycosyltransferase.
The first sugar in an inhibitor of the invention may be a monosaccharide or disaccharide, preferably a monosaccharide. Examples of these sugars include galactose, glucose, mannose, ribose, fructose, deoxyribose, preferably ribose and deoxyribose. The first sugar may be modified for example, the hydroxyls may be blocked with acetonide, acylated, or alkylated or substituted with other groups such as halogen.
The first sugar may be part of a nucleoside namely guanosine, adenosine, thymidine, cytidine or uridine, preferably uridine. A heterocyclic amine base in a nucleoside may be modified. For example, when the base is uridine it may be modified at the C-5 position with groups including but not limited to alkyl or aryl with electron donating and electron withdrawing groups.
The second sugar is selected based on the type of glycosyltransferase to be inhibited, and it is typically D-GlcNAc (see Table 16).
The ion may be any counter-ion including sodium, lithium, potassium, calcium, magnesium, manganese, cobalt ions and the like, as well as nontoxic ammonium, quaternary ammonium, and amine cations, including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like.
In a preferred embodiment of the invention an inhibitor is prepared comprising UDP-GlcNAc complexed with an ion, wherein the orientation of the linkage between the uridine deoxyribose phosphate and GlcNAc is synclinal, and the phosphorous-oxygen bonds linking the phosphates of the diphosphate (xcexa82, xcexa83) are in synclinal, and -anticlinal or antipleriplanar orientation, or a functional derivative thereof.
The term xe2x80x9cfunctional derivativexe2x80x9d is intended to include xe2x80x9cvariantsxe2x80x9d xe2x80x9canalogsxe2x80x9d or xe2x80x9cchemical derivativesxe2x80x9d of the inhibitors. The term xe2x80x9cvariantxe2x80x9d is meant to refer to a molecule substantially similar in structure and function to an inhibitor or a part thereof. A molecule is xe2x80x9csubstantially similarxe2x80x9d if it has a substantially similar structure or it possesses similar biological activity. The term xe2x80x9canalogxe2x80x9d refers to a molecule substantially similar in function to an inhibitor of the invention. The term xe2x80x9cchemical derivativexe2x80x9d describes a molecule that contains additional chemical moieties which are not normally a part of the base molecule, or one or more of the atoms is optionally replaced by another atom.
The inhibitors of the invention may be useful for the prevention, treatment and prophylaxis of tumor growth and metastasis of tumors; the prevention of tumor recurrence after surgery; the treatment of other anti-proliferative conditions such as viral infections; the stimulation of bone marrow cell proliferation, the treatment of immunocompromised patients, such as patients infected with HIV, or other viruses or infectious agents including bacteria and fungi; the prevention and treatment of diseases caused by bacterial pathogens having carbohydrate structures on their surface associated with virulence such as Neisseria, Haemophilus, E. coli, Bacillus, Salmonella, Campylobacter, Klebsiella, Pseudomonas, Streptococcus, Chlamydia, Borrelia, Coxiella, Helicobacter, and Mycobacterim species; or, the treatment of inflammatory disorders such as asthma, rheumatoid arthritis, inflammatory bowel disease, and atherosclerosis. The inhibitors may also be used in patients undergoing bone marrow transplants, and as hemorestorative or chemoprotective agents in patients with chemical or tumor-induced immune suppression.
The inhibitors may be converted using customary methods into pharmaceutical compositions. The pharmaceutical compositions contain the inhibitors either alone or together with other active substances. Such pharmaceutical compositions can be for oral, topical, rectal, parenteral, local, inhalant, or intracerebral use. They are therefore in solid or semisolid form, for example pills, tablets, creams, gelatin capsules, capsules, suppositories, soft gelatin capsules, liposomes (see for example, U.S. Pat. No. 5,376,452), gels, membranes, and tubelets. For parenteral and intracerebral uses, those forms for intramuscular or subcutaneous administration can be used, or forms for infusion or intravenous or intracerebral injection can be used, and can therefore be prepared as solutions of the inhibitors or as powders of the inhibitors to be mixed with one or more pharmaceutically acceptable excipients or diluents, suitable for the aforesaid uses and with an osmolarity which is compatible with the physiological fluids. For local use, those preparations in the form of creams or ointments for topical use or in the form of sprays should be considered; for inhalant uses, preparations in the form of sprays should be considered.
The pharmaceutical compositions can be prepared by per se known methods for the preparation of pharmaceutically acceptable compositions which can be administered to patients, and such that an effective quantity of the active substance is combined in a mixture with a pharmaceutically acceptable vehicle. Suitable vehicles are described, for example, in Remington""s Pharmaceutical Sciences (Remington""s Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., USA 1985). On this basis, the pharmaceutical compositions include, albeit not exclusively, the inhibitors in association with one or more pharmaceutically acceptable vehicles or diluents, and contained in buffered solutions with a suitable pH and iso-osmotic with the physiological fluids.
The inhibitors may be indicated as therapeutic agents either alone or in conjunction with other therapeutic agents or other forms of treatment (e.g. chemotherapy or radiotherapy). The inhibitors can be used to enhance activation of macrophages, T cells, and NK cells in the treatment of cancer and immunosuppressive diseases. By way of example, the inhibitors may be used in combination with anti-proliferative agents, antimicrobial agents, immunostimulatory agents, or anti-inflammatories. In particular, the inhibitors may be used in combination with anti-viral and/or anti-proliferative agents, such as Th1 cytokines including interleukin-2, interleukin-12, and interferon-xcex3, and nucleoside analogues such as AZT and 3TC. The inhibitors may be administered concurrently, separately, or sequentially with other therapeutic agents or therapies.
The compositions containing inhibitors can be administered for prophylactic and/or therapeutic treatments. In therapeutic applications, compositions are administered to a patient already suffering from a disease or condition as described above, in an amount sufficient to cure or at least alleviate the symptoms of the disease and its complications. An amount adequate to accomplish this is defined as a xe2x80x9ctherapeutically effective dosexe2x80x9d. Amounts effective for this use will depend on the severity of the disease, the weight and general state of the patient, the nature of the administration route, the nature of the formulation, and the time or interval at which it is administered.
In prophylactic applications, compositions containing inhibitors are administered to a patient susceptible to or otherwise at risk of a particular disease. Such an amount is defined to be a xe2x80x9cprophylactically effective dosexe2x80x9d. In this use, the precise amounts depend on the patient""s state of health and weight, the nature of the administration route, the nature of the formulation, and the time or interval at which it is administered.
Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the invention are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.